CN115487539B - Direct capture using large bead chromatography media - Google Patents

Abstract

A continuous process in which subsets of a plurality of mutually identical columns are connected in series. A process liquid, such as crude cell culture harvest, is supplied to the uppermost You Zhu of the subsets, continuously flows through the series-connected columns and through the lowermost free subset and into a downstream collection vessel. Once the packed bed of the most upstream column is saturated with product, that column is disconnected from the subset and removed from the series connection, and a replacement identical column is added so that it is connected in series downstream of the most downstream column of the subset, and the process is repeated.

Description

Direct capture using large bead chromatography media

Technical Field

The present invention relates to the direct or primary capture (e.g. capturing of product from a crude feed) of product present in a preferably uncleaned (i.e. unpurified) process liquid using a column for liquid chromatography (e.g. radial flow) comprising a packed bed of large beads (large beads). In particular, the invention relates to downstream processing of biological products from cell cultures or cell fermentation harvests, and to related liquid chromatography systems.

Background

General background on chromatography

It is often necessary or desirable to fractionate the fluid mixture to separate or isolate useful or desired components. This can be achieved by using a liquid chromatography system.

Chromatographic systems of various sizes are used in laboratory analysis operations and industrial scale production operations, where separation steps such as fractionation from human blood or capturing or removing impurities from drugs can be performed on a large scale in batch processes and now also in continuous processes.

Liquid chromatography can be briefly described as fractionating the components of a mixture based on differences in the physical or chemical properties of the components. Various liquid chromatography systems use fractionation or solid matrices to fractionate components. Some liquid chromatography matrix systems fractionate the components of a mixture based on physical parameters such as molecular weight. Still other liquid chromatography systems will fractionate the components of a mixture based on chemical criteria such as ionic charge, hydrophobicity, and the presence of certain chemical moieties (such as antigenic determinants or lecithin binding sites on the components), which together are characterized as "affinity" moieties.

Liquid chromatography typically uses a separation column. The separation column comprises a stationary phase, packing, packed bed or matrix medium or material, as known in the art, which interacts with the various components of the sample fluid to be separated. The composition of the separation medium depends on the fluid being directed therethrough in order to produce the desired separation.

In order to produce a chromatographic column capable of effectively separating biological substances, it is common to pack the fine particles of the separation material as tightly and uniformly as possible in a column tube, packed bed. The packing of the column, or column packing as commonly referred to, is typically achieved by closing one end of the column with an outlet device comprising a filter element and pouring or pumping a liquid suspension of particles under pressure into the column from the other end of the column. Although the pumped liquid is able to pass through the filter element substantially unimpeded, the particles are retained by the filter element, thereby constituting a bed of particles along the length of the tube. When filling the column tube, the particles are pressed out towards the tube wall and the particle bed obtains a stable compacted state, wherein the particles are well distributed by the pressure generated by the compression of the pump or top filter, which state is maintained throughout the filling process.

The first type of separation column generally known in the art is a cylindrical structure and the fluid flows axially through a bed of separation medium (packing or matrix) retained in the column. The media bed is retained between supports or filter plates (frames) on one or both ends of the column. As the sample (also referred to as "feed") or elution (also referred to as "desorb") fluid passes through the bed of separation medium, the components of the associated fluid travel at different rates due to their different interactions with the matrix or packing material. As a result, these components separate out (i.e., have different elution times) in the outlet stream of the column.

With the need for high performance low pressure chromatography, horizontal or radial flow chromatography columns have been developed. Such horizontal or radial flow columns are described and claimed, for example, in U.S. patent nos. 4,627,918 and 4,676,898. In a horizontal or radial flow type column, the sample/adsorption and elution/desorption fluids are introduced via a distributor to the outer or circumferential wall or surface of a separation medium or matrix comprised of packing materials, where the components are separated, and the fluids pass horizontally or radially inward through the separation medium to a center or collection port, and then elute from the column at different rates at different times. This horizontal flow column design has a high cross-sectional area and a very low effective bed height. Thus, it provides the ability to handle very high flow rates at low operating pressures.

As used herein, the term "horizontal or radial flow pattern" is interchangeably defined as the flow of a sample (e.g., a biomolecule) or eluent or washing fluid through a chromatography column in a direction perpendicular to the longitudinal axis of the column, regardless of the position of the column relative to a table or support or other device for supporting or stacking the column.

This horizontal mode of chromatographic separation can be achieved by a chromatographic column configured with an inner ring and an outer ring with matrix material filled between the inner and outer rings, thereby having a torus or annular shape. Thus, the bed height is calculated as the distance between the inner and outer rings. The chromatography is thus carried out radially in the column.

This horizontal mode of column configuration results in a uniform bed height because the inlet and outlet distributors are fixed. The distributor and collection channels are designed to provide uniform application of the sample and horizontal flow lines through the chromatographic bed. Long, vertical, column assemblies with horizontal flow are easy to manufacture, easy to package and handle. Furthermore, since the bed height is constant along the column length, the cross-sectional area and the bed volume are both proportional to the column length. Thus, by linearly increasing the length of the column in proportion to the desired scale of operation, scale up is possible. At any scale of operation, the pressure drop remains constant and scale-up is readily achieved by linear increases in bed length.

The horizontal mode column is particularly useful for high performance low pressure chromatography used in conjunction with separation of biomolecules such as proteins or other organic or inorganic compounds that are particularly sensitive to shear forces. This column type can be used for high performance and conventional chromatography, identification and separation of mixtures in a preparative mode, and also provides a scale-up to larger columns for separating components from mixtures in amounts suitable for industrial or production purposes.

The development of chromatographic columns aims to provide ease of operation and various additional benefits of particular commercial importance. These include: (a) the ability to be sterilized by autoclaving; (b) Improved hygiene due to the design features of the product with less carryover from one batch to the next; (c) resistance to solvents; (d) food-grade FDA compliant material; (e) improved pressure resistance; (f) low cost; (g) potential for complete or partial automation; (h) the ability to provide a disposable column; (i) linear process upgrades.

During use, the performance of the packed bed, i.e. the beads, is continuously degraded. Since beads are usually very expensive (about 1-20 euros/ml), the packed bed should be completely exhausted, at least for industrial applications. Preferably, the same packed bed is used multiple times (practically limited to 200 times) and thoroughly cleaned before each next time (i.e., between each two successive uses). Although very careful, due to aging, the theoretical maximum number of uses does not match very much in the case of use over a period of many months (e.g. 6 or 12 months). Aging is exacerbated because any potential contaminants are inevitably not completely removed during cleaning between subsequent uses.

Specific background of direct capture

Now, more direct capture specific information is provided by mammalian cell culture as an illustrative example of biomolecules.

Bio-pharmaceutical products, typically therapeutic proteins, can be produced by a variety of organisms. The most common production method at present is mammalian cell culture, where microorganisms grow and are designed to produce additional specific compounds that are not normally produced by the (e.g. CHO) cells. The therapeutic product designed is the target active pharmaceutical ingredient (also known as an API). Because many biological compounds carry the characteristics of a (non-human) host, the products produced by non-human cells can cause rejection by recipients where the cell culture cells are "humanized". This is accomplished by fusion with mammalian cells to produce a human trait, and is referred to as mammalian cell culture. Mammalian cells are grown in large vessels with carefully balanced energy-cocktail, medium, supporting their growth. The medium contains all nutrients to obtain an exponential cell growth. When the cells are well programmed, they will produce the compound of interest in large amounts, and when possible, the API is secreted into the surrounding medium (extracellular). Typical cell culture volumes for API production are 1,000-10,000 liters.

The disadvantage of these perfect small designs is that the fused cells are more fragile. Cells must be gently treated to prevent rupture (i.e., they must remain viable). Broken cells cause two problems: cells stop production, but more importantly their internal substances are secreted into the culture medium. Host Cell Proteins (HCPs) and host cell DNA contaminate the medium, which makes purification more difficult, and digestive enzymes from the internal cells begin to digest all proteins in the medium, i.e., also the target API. Gentle mixing and even uniform air sparging can cause cell disruption during cell culture. When the cell culture reaches its growth limit, the culture is harvested. Harvesting 1,000-10,000 liters of cell culture in a short period of time without damaging the cells is very challenging.

The prior art on an industrial scale is rapid clarification, centrifugation and/or filtration of cells, separating cell clusters from the surrounding medium (plasma) containing the API. Unfortunately, centrifugation and filtration also lead to cell disruption, contaminating the plasma. An unclarified/crude, e.g., a device that does not filter and/or centrifuge the process liquid of the column feed.

Several decades ago, expanded Bed Adsorption (EBA) has been invented to provide for the treatment of crude feed without purification. This process is sometimes referred to as "fluidized bed adsorption". The cell culture was fed to the bed by precipitating the bottom of the settled chromatography bead slurry. There is no restrictive screen or other restriction above the settling beads and the bed is allowed to expand upward by the force of the upward flow. The column was very high and when precipitated, the bed was allowed to expand freely, only about 1/3 of the packed with precipitated beads. However, no resistance results in a preferential path, and fragments of accumulated cells stagnated thereby result in contamination outside the path. To prevent this, the chromatography beads were manually made heavier by including a metal core. The precipitated beads are thus heavier, creating a density (resistance), i.e. a dynamic pore size reduction, forcing the cells to be evenly distributed over the whole surface of the bottom and forcing the beads to leave room for passage. While this technique works well in some cases, it is difficult to control and is often not very robust.

Related background Art

US5466377a (graphics et al, published 1995) discloses the use of large bead chromatography particles in standard or conventional low pressure packed bed chromatography columns for direct capture of products from unclarified process liquids as a system for downstream processing of biologicals from different types of homogenized cell cultures, microbial cell cultures or bacterial cell fermentation harvests, using microorganisms about 0.5 to 1 micron and at least 10 times smaller than mammalian (CHO) cells. The use of an axial column of diameter 1.5 cm is disclosed, having an end plate screen of large holes (60-180 microns, equal to 0.06-0.18 mm holes), beads of diameter 100-300 or 300-500 or 500-800 or 800-11000 microns (equal to 0.1-0.3 or 0.3-0.5 or 0.5-0.8 or 0.8-1.1 mm respectively) and a bed volume of 9 ml. No further information about the column is provided. However, from the column diameter and volume data provided, the "bed height", i.e., the axial distance between the end plate screens, can be calculated and is equal to 5.0 cm. At such small scale operations in a laboratory environment, there are typically no problems mentioned herein, such as concerns about contamination, aging, and complete exhaustion of the packed bed for economic reasons. However, attempts to produce large-scale at an economic level have shown that even with the homogenates and much smaller bacterial cells as shown, the axial column type is not suitable for direct capture industrial applications.

Disclosure of Invention

The object of the present invention is general and according to a first aspect an improved, pollution-free, low-pressure, gentle-acting packed bed chromatography column is provided, packed with large beads for efficient direct capture of products (e.g. biomolecules) from unclarified (or crude) process liquids, and related processes, in particular for downstream processing of biologicals or similar cell cultures or cell fermentation harvest from mammalian CHO (cell culture densities typically higher than 0.1 or 0.5 or 1 or 2 and/or lower than 50 or 100 million cells/ml). In a second aspect, the first downstream processing step, i.e. the primary capture of the high density cell culture harvest, is in a radial flow packed bed capture column, directly from the cell culture vessel without any intermediate, filtration or other clarification equipment, nor any harvest retention step or retention vessel. The treated cell culture fluid (the processed cell culture liquid, depleted from the product of interest) depleted from the product of interest exits the primary capture column (typically containing cell/particle content) directly into, for example, a waste or collection vessel. By removing the filtration device, a reduction in capital costs and setup time is achieved, while removing the harvest retention vessel reduces the available incubation time for proteolytic attack and automatic digestion of the product of interest by host cell enzymatic activity during the residence time of the product of interest in the vessel, so removing the vessel will reduce the risk of product damage or digestion. It is also an object to avoid cell damage. Other objects can be found in other parts of this disclosure.

According to the present invention, a solution is provided by applying a high performance chromatography column characterized by a packed bed and a horizontal or radial flow through the packed bed. This direct capture technique using radial flow is also referred to herein as ctrc (cell tolerance radial affinity chromatography). It should be understood that columns designed for radial flow generally mean that the packed bed is annular or ring-shaped, although alternative designs are also contemplated, preferably designs that mimic the flow conditions of radial flow columns, such as wedge-shaped or conical, wherein one filter plate has a larger surface area (I) than the other (O) to meet functional I/O requirements. Typically, the filter plate through which the liquid to be treated enters the packed bed, also referred to as the inlet filter plate, has a larger surface area than the filter plate through which the liquid exits the packed bed, also referred to as the outlet filter plate. Thus, the liquid to be treated generally flows radially inwardly from the inlet filter plate to the outlet filter plate. A typical upstream holding vessel can also be avoided so that liquid from the cell culture can be fed directly to the chromatographic column.

Surprisingly, the inventors have found that a properly designed, properly balanced radial column can create additional dynamic resistance that is required to uniformly distribute the liquid over the entire surface area without reducing the pore size. The ctac radial column is characterized by an inlet surface area that is greater than an outlet surface area. The packed bed is radial, wedge-shaped or conical. The packing density of the packed bed is suitably controlled by the annular packing method resulting in an even distribution of bead porosity throughout the packed bed. The interstitial volume between the filled beads is the same at the inlet (larger surface) compared to the outlet (smaller surface). Thus, the gap pore size is constant throughout the column. Furthermore, no purge and/or harvest hold steps are required before the column and during the flow of the process liquid through the column, the liquid (typically the cell/particle content) exiting the column can be fed directly into, for example, a waste collection vessel. If a treatment such as high density continuous perfusion of a cell culture, wherein cultured cells e.g. remain in the cell culture upstream of the column, results in a cell culture with extreme cell density, and wherein only a relatively small volume containing a limited amount of cell material is continuously sent to downstream treatment, the column may be fed (e.g. connected) directly from/directly to the cell culture outlet, wherein the feed is optionally (partly) recycled back into the upstream source (e.g. continuous perfusion of the cell culture) by means of the effluent, or the column output is sent e.g. into a waste collection vessel.

This solution is surprising, since passing cells (typically 5-20 microns) through a packed bed without the above-mentioned problems (e.g. clogging, contamination, economy of use) is always considered impossible for industrial or rational use. On the one hand, larger beads fill into a homogeneous matrix, leaving "open spaces", i.e. interstitial volumes, between them, which is beneficial. Through this interstitial volume, the cells can flow with limited or even no obstruction (drag, shear). A low or even lack of resistance is beneficial for cell viability, with cells flowing smoothly between beads towards the outlet of the column with little or no obstruction. The driving force for cell travel is the flow of liquid. However, low or even no resistance also means a preferential path, cell accumulation and a tendency to contaminate debris and complete obstruction, as in the earlier EBA examples.

Similar and in parallel to the earlier EBA examples, the main challenge in the application of packed bed chromatography is to force the incoming liquid to be evenly distributed over the whole surface, thus preventing preferential paths without creating pore size reduction, as in packed beds this will be static and will lead to immediate column plugging. In the case of earlier reported attempts to use a conventional (axial) chromatographic column with packed beds, the column will plug and/or contaminate.

Without being bound by theory, it is believed that the surprising effect of the present invention can be explained by the Kozeny-Karman equation (i.e., besselink: are axial and radial flow chromatography different. According to this equation, an increase in apparent velocity results in an increase in resistance to flow between beads. Furthermore, the increase in apparent velocity appears to be proportional to the I/O ratio (sometimes referred to as "alpha" or "alpha"), which is directly related to the shape of the radial column.

The decrease in surface area towards the outlet of the radial flow pattern column results in an increase in local flow velocity, which in turn results in an increase in resistance without decreasing the interstitial space, which in turn results in a uniform distribution of liquid over the entire surface. Although the surface at the outlet is smaller, focusing the liquid flow like an optical lens, the uniform distribution is transferred to the larger surface at the inlet. Instead of radial inward flow, radial outward flow through a packed bed may be applied to the column of the present invention.

Another problem that does not exist in the case of EBA but does exist in the case of packed bed columns is the screens (filter plates) between which the bed is packed and which separate the internal volume from the external flow path. Experiments have shown that only very thin and open/smooth structured filter plates allow the cells to pass without rupture or causing differential damage.

The thin and open-structured filter plates of the preferred radial flow column allow for excellent cell viability. The cylindrical form of the radial posts allows the filter plate to be thinner because the cylindrical object has many times the shape stability of a planar object having the same material thickness. Thinner filter plates result in greater viability and fewer ruptured cells.

Bead size, bead rigidity, packing density, inlet surface area/outlet surface area ratio (I/O ratio) and volumetric flow rate are now some possible parameters for supporting a suitable dynamic resistance for a uniform distribution of the liquid to be treated. The proper balance between these parameters must be optimized for each individual application. The resulting dynamic resistance supporting an even distribution is disadvantageous for the application.

Preferably, for the filter plate, one or more of the following applies: made of stainless steel; electropolishing the surface; a hydrophilic surface; a layer or sheet comprising at least or exactly one or two or three or four stainless steel woven filaments directly superimposed on each other, preferably at least one sheet according to a plain weave (plain weave) or twill weave (twilled weave) or plain dutch weave (plain dutch weave) or twill dutch weave or reverse plain dutch weave or reverse twill dutch weave or five-harness weave pattern, such as a filtration layer; these sheets provide a joint assembly, preferably sintered to each other (also known as diffusion bonding); at least one or two, e.g., each, of the sheets is woven from wire having a diameter that differs from the immediately adjacent sheet by at least 10% or 20%, e.g., greater or lesser; the thickness of the lines between the sheets increases from one surface of the filter plate to the other, preferably from the surface facing and/or bounding the packed bed; the sheet has a line thickness of at least 25 or 50 microns (equal to 0.025 and 0.05 mm) and/or no greater than 500 microns (equal to 0.5 mm); at least one or two, e.g., each, of the sheets have a pore size that differs from the immediately adjacent sheets by at least 10% or 20%, e.g., greater or less; the pore size between the sheets increases from one face of the filter plate to the other, preferably from the surface facing and/or bounding the packed bed; the sheet (e.g. reinforcing sheet) having the coarsest lines and/or the largest pore size (e.g. at least 500 microns (0.5 mm)) is the preferred final inner or outermost sheet of the filter plate, preferably the sheet furthest from the packed bed; a thickness of at least 0.3 or 0.8 or 1.0 mm and/or not more than 1.2 or 1.5 mm; a pore size (this is the "nominal" pore size, defined by the diameter of the largest rigid sphere that is capable of passing through the pore) of at least 50 or 100 and/or no greater than 200 or 500 microns (equal to 0.05, 0.1, 0.2 and 0.5 millimeters, respectively); preferably by continuous weld to the axial end plates; the number of sheets of the inner filter plate is one or two or three more than the number of sheets of the outer filter plate; comprises a single filter layer; the filter layer is directly exposed to the packed bed; on the side of the filter layer facing the packed bed, there is no layer, for example a protective layer; the filter layer provides a surface layer; there is no layer or only a protective layer on one side of the filter layer and only a protective or dispersing layer on the other side, and possibly exactly one or two further layers, preferably reinforcing layers.

Preferably, the I/O ratio is at least 1.5:1 or 2:1 or 2.5:1 and/or not greater than 3:1 or 3.5:1 or 4:1 or 5:1 or 10:1.I/O is the ratio between the radii (shown by arrows R1 and R2 in fig. 1) of the outer and inner filter plates of the column.

The bed height is preferably at least 10 or 20 and/or not more than 100 or 150 or 200 or 300 mm. The bed height (shown by arrow H in fig. 1) is the distance between the inner and outer filter plates, or in other words, the difference between the diameters of the inner and outer filter plates. The bed volume is preferably at least 10 or 25 or 50 or 100 or 200 or 250 or 500 ml and/or not more than 10 or 20 or 150 or 250 liters.

The diameter (in millimeters) of the inner filter plate is preferably: at least 10 or 15 or 20 and/or not more than 80 or 100 or 150 or 300, such as 23 or 50 or 60 or 100.

Alternatively, instead of a complete annular or ring-shaped bed for containing beads, a section thereof is used (i.e. fig. 13).

In a typical cylindrical space defined by the inner filter plate, a core member is preferably provided, the outer wall of which defines an inner flow channel with the inner filter plate.

Preferably, the outer flow channels defined outside the outer screening deck have a width of at least 0.5 mm and/or not more than 2 or 3 or 5 or 10 mm. Preferably, the inner flow channel has a width of at least 0.5 mm and/or not more than 2 or 3 or 5 or 10 mm, more preferably equal to the outer flow channel width times the I/O ratio (also referred to as "I/O-width") and/or a width at least 0.5 or 1 or 2 and/or not more than 5 or 10 mm wider than the outer flow channel width. In the case of a tapered core member, the internal flow channel width is preferably at least 0.5 and/or no more than 1 or 2 millimeters at one axial end and at least 1 or near I/O width and/or no more than 3 or 4 or 6 millimeters at the opposite longitudinal end (the "longitudinal" direction being parallel to the axial direction of the column) and/or at least 1 or 2 and/or no more than 3 or 5 or 10 millimeters wider than the narrow axial end.

Preferably, the invention is applied to a continuous process wherein two, three or more, for example five, preferably subsets of mutually identical columns are connected in series, preferably no more than 5 or 10. For this process, the number of columns required for a complete set is equal to the number of columns of the subset plus one, two, three or more columns, preferably the same as the columns of the subset. A process liquid, such as crude cell culture harvest, is supplied to the uppermost You Zhu of the subsets, flows continuously through the series-connected columns, and, for example, the culture and cells depleted from the product of interest, pass through the lowermost free subset, and into a downstream collection vessel, such as a waste collection vessel. Once a sufficient or predetermined amount of time has elapsed, for example, the time that the packed bed of the most upstream column has captured a predetermined amount of product, for example, becomes full of product, the most upstream column is disconnected from the subset and removed from the series connection, and a replacement identical column, preferably containing a new or reset packed bed, is added so that it is connected in series downstream of the most downstream column of the subset, and the process is repeated. During the time required to saturate the most upstream column of the subset with target from process liquid, the saturated column that was most recently disconnected from the subset is treated off-line to reset the packed bed (e.g., wash, elute/desorb, purge, and prepare bed) to become available for addition to the subset to become saturated again. Columns with low flow resistance allow this off-line process to be performed at much higher speeds, possibly up to 10 times the flow rate through the subset. This results in a time lapse, resulting in a reduction of the minimum number of columns required and thus in a reduction of the total volume of the complete plant. This cycle is repeated a number of times in order to repeatedly remove the most upstream column from the subset of serially connected columns after the time has elapsed and to add the most downstream column to the subset of serially connected columns so that the number of columns of the subset remains the same during the whole process. Typically, the columns removed from the series connection have their packed beds treated, e.g. cleaned, to become reset and thus adapted to return to the series connection, initially as the most downstream column, and then to continuously obtain an upstream ordering as other reset columns are added downstream. The reset process preferably starts and completes within the time that elapses between the removal of two consecutive pillars from the series connection, so that the pillars are removed from the series connection only as briefly as possible and the smallest possible number of pillars is required. Preferably, the number of columns as part of the continuous process is the number of columns connected in series (as a subset) plus a single column in the reset process plus possibly one or two spare columns.

The columns of the subset are preferably interconnected such that the process liquid flows through the packed bed in the same direction (i.e. radially inward or outward) for all columns, but in the alternative at least one column has its packed bed flowed in the opposite direction to at least one or two other columns of the subset. For example, in a subset, the most upstream column has its packed bed flowed in a radially inward direction, while the more downstream column in the same subset has its packed bed flowed in a radially outward direction.

For the same volumetric yield of captured product, continuous processing requires a smaller volume of beads, e.g., at least 25% or 50% or 75% less, than batch processing.

The packed bed volume of each column associated with the continuous process is preferably at least 25 or 100 or 250 milliliters and/or no greater than 10 or 20 or 100 liters.

The purpose of such continuous treatment is to significantly reduce the ecological footprint, installation time, treatment time, waste, time and space spent in clean rooms.

The column, preferably a subset of the continuous processes, may be used to remove contaminants such as growth rate limiting contaminants from the process liquid, wherein the contaminants are the target of the packed bed and the liquid leaving the column or subset is used, for example, as a medium for feeding the cell culture.

For continuous processing, it is very important that the process liquid leaving the further upstream column is still free of contaminants, e.g. damaged cells or cell contents, such as proteins or DNA. This may be provided by designing the outlet filter plate such that cells can pass through it intact. For continuous and batch processes, the inlet and/or outlet filter plates are preferably designed so that cells can pass through it intact.

The beads preferably have a diameter of at least 200 and/or not more than 500 or 1000 micrometers (equal to 0.2 and 1.0 mm) and/or are hydrophilic.

In one embodiment, the present invention relates to a liquid chromatography column utilizing a horizontal or radial flow of sample material therethrough, comprising one or more of the following: a housing defining a chamber therein and including at least one preferably removable axial/longitudinal end; first (outer) and second (inner) axially/longitudinally extending porous filter plates or membranes located within the chamber of the housing; a bed or packing of chromatographic separation material, preferably in particulate form, located within said chamber of said housing and intermediate said porous filter plates, a first or outer of said porous filter plates being adjacent to the housing wall and defining with said wall a cylindrical annular outer flow passage, a second or inner of said porous filter plates being adjacent to the optional core member and defining with said core member a cylindrical annular inner flow passage; dispensing means operatively connected to said external flow passage; collecting means operatively connected to said internal flow passage; a supply channel (also referred to as a liquid inlet) operatively connected to the dispensing means, and a discharge channel (also referred to as a liquid outlet) operatively connected to the collecting means; the distribution device and the external flow channels are configured to uniformly direct the relevant material to be separated in the bed in a substantially horizontal direction across the axial/longitudinal length of the bed.

Furthermore, the porous filter plates are coaxially positioned relative to each other. In practice, the first porous filter plate has a larger cross section than the second porous filter plate and if the core member is applied, the core member is located in the centre of the housing chamber, coaxially with the first and second filter plates.

Preferably, one or more of the following further features are applicable to the column of the present invention: one of the axial ends is penetrated by a supply channel (or liquid inlet) and a discharge channel (or liquid outlet); the drain channel is coaxial with the first and/or second filter plates; the outer flow passage is radially spaced from the housing center and/or the discharge passage; the housing has a substantially cylindrical wall; the first and second filter plates are part of a cartridge removably received in the housing; at one axial end, the first and second filter plates are connected by an end wall closing the space between the first and second filter plates at said axial end, and possibly having a passage for a drain channel or core member, and/or at the opposite axial end, the internal flow channel is closed by an end wall connected to the second filter plate; the outer and/or inner flow channels extend substantially the entire height of the chamber; in the operative position within the chamber, the packed bed extends beyond the axially extending housing wall from an end opposite the axial end wall; an outflow channel connected to one or more of the internal flow channel, the collecting space, the discharge channel, extending inside the core member, preferably in the longitudinal direction of the core member, and/or flowing out at the axial/longitudinal ends of the core member within the chamber; the core member extends substantially the entire height of the chamber; the core member penetrates the axial end portion; the core member protrudes from the axial end portion; the axial end portion has a central bore penetrated by the core member; the core member maintains a gap with its axial/longitudinal free end and an associated axial end and/or tapers to a narrow dimension towards said axial end; the dispensing device comprises a radially outwardly narrowing portion, preferably the core member surrounds a dispensing space, preferably between the axial end section and the space containing the filler, to which the external flow channel is preferably connected, more preferably leads; preferably due to the tapering of the core member, the cross-sectional area of the internal flow channel preferably increases continuously along the axial direction; one axial end, preferably associated with the axial free end of the core member, contains means, such as a closable filling port, to provide a filling material for the space between the filter plates for column filling purposes, while the column is fully assembled (e.g. a filling system according to WO2007136247 (raeds)). There is a seal between the core member and at least one of the axial end and the cartridge or end wall connecting the first and second filter plates; the column has a convenient external size of about one or five or ten litres capacity, possibly even up to a hundred litres.

Preferably, the present invention relates to one or more of the following: a homogeneous packed bed matrix of adsorbent beads having a defined bead size to produce a porosity-controlled matrix throughout the packed bed such that it is capable of passing the concentrated unclarified mammalian (or any other) cell culture harvest unimpeded; the cell diameter of the cell culture harvest is about 0.1-10% of the bead diameter; the average bead diameter is about 120-500 μm; CHO cells are typically about 5-15 or 20 μm; the uniform distribution of the cells of the cell culture within the porous matrix is produced by a concentration of flow caused by an increase in resistance, as explained by the Kozeny Carman equation, as represented by the radial packed bed; the increase in velocity of the radial packed bed column traveling from the inlet to the outlet, or the decrease in velocity when traveling in the opposite direction, creates a resistance that drives the concentrated/uniform distribution of cells without reducing the porosity between the bead matrices within the packed bed; multi-column (semi-) continuous operation, wherein subsets of columns connected in series are continuously loaded with cell culture harvest.

When handling and processing e.g. high density "continuous perfusion" of cell cultures, most of the culture cells remain in the cell culture upstream of the column, resulting in cell cultures with extreme cell densities, and wherein only a relatively small volume containing a limited amount of cell material is continuously sent to downstream processing. The column may be fed directly, e.g. connected, to a cell culture outlet, wherein preliminary capturing of the product is selected while the column effluent is sent to a collection vessel or feed is (partially) recycled back to the upstream source via the effluent, alternatively the column may be filled with a large bead adsorbent designed to capture/remove contaminants, e.g. limiting the growth rate, from the continuously perfused cell culture while the clean medium is recycled back into the cell culture.

When used for primary capture, the column is replaced with a clean column to allow recovery of the product. Alternatively, the saturated column that captures the contaminants may be replaced with a clean column to allow cleaning of the contaminated column.

Thus, the process is also designed as a continuous process.

Drawings

The accompanying drawings, which are incorporated in and form a part of the specification, illustrate embodiments of the present invention and, together with the description, serve to explain the principles of the invention. The following are shown:

FIGS. 1-3 are cross-sectional side views of examples of posts;

FIG. 4 is a top view of the column of FIG. 3;

FIG. 5 is a perspective view of another post with portions cut away from below;

FIGS. 6 and 13 are two examples of a continuous process in which a subset of three columns in series are applied;

7a, 7b, 7c, 7d, 8a, 8b and 8c show the weave pattern of the filter ply;

FIG. 9 is a perspective exploded view of a prior art filter plate;

FIGS. 10-11 are cross-sectional side views of prior art filter plates;

FIG. 12 is a perspective view of a ring filter bed;

the following reference numerals are used: column 1; a cylindrical housing wall 2; an axial housing end plate 3; a first seal 4; a liquid inlet 5; a liquid outlet 6; a packed bed 7; an internal flow passage 8; a packed bed filling opening 9; a connector 10; a filling pipe 11 for a packed bed; a second seal 12; a third seal 13; an external flow channel 14; a core 15; an inner filter plate 16; an outer filter plate 17; an axial bed end plate 18; a distribution space 19; a collector space 20; an outflow channel 21; a liquid outlet 22; bead height H; an outer filter plate radius R1; an inner filter plate radius R2; axial arrow a (fig. 3). The radial direction is perpendicular to the axial direction.

Detailed Description

Each of the liquid chromatography columns shown in fig. 1-5 includes: a cylindrical housing defining a chamber therein and comprising a circular removable axial end plate 3; a cylindrical first (outer) porous filter plate 17 and a second (inner) porous filter plate 16 or membrane; a bed 7 or packing of particulate chromatographic separation material positioned intermediate the porous filter plates; optionally an axially extending core 15. The axially extending cylindrical housing wall 2, the first and second filter plates 17, 16 and the core 15 are coaxial.

The first or outer filter plate 17 is adjacent to the axially extending cylindrical housing wall 2 and together with said wall defines a cylindrical annular outer flow channel 14, for example 0.5 mm wide. A second or inner filter plate 16 is adjacent to the core member 15 and defines with said core member a cylindrical or conical annular inner flow channel 8, for example 1.0 mm wide. In fig. 1, the core member 15 is absent.

In one embodiment, the axial top end plate 3 is penetrated by both the supply channel 5 and the discharge channel 6. These channels are coaxial with the filter plate.

The first and second filter plates may be part of a cartridge removably contained in the housing.

At both axial ends, the first and second filter plates are connected by a radially extending end wall 18, thereby closing the space between the first filter plate 17 and the second filter plate 16 to capture the annular packing providing the filter bed.

The filter bed 7 plus the core 15 almost completely fills the housing 1. Between the top surface 18 of the filter bed 7 and the bottom surface of the axial end plate 3 there is a distribution space 19 into which the external flow channels 14 open. The distribution space may taper in a radially outward direction and merge with the circumferentially extending outer flow channel 14 at the radially outer circumference thus narrowed. The circumferential internal flow channel 8 surrounding the core 15 tapers in the axial direction from one axial bed end plate 18 to the opposite axial bed end plate along the core body 15 and merges at the lower end of the core with a collecting space 20 defined between the lower end of the core and the bottom end 3 or end wall 18 of the housing. This conical shape of the inner flow channel 8 provides a wide and narrow axial end due to the axial taper of the core. The tapering of the distribution space 19 and the inner flow channel 8 optimizes the flow characteristics. The outflow channel 21 extends longitudinally through the core 15 (fig. 2) and is connected to the liquid outlet 6 and the collecting space 20.

Fig. 3 shows an embodiment in which a part of the liquid entering through the liquid inlet 5 leaves the column 1 without passing through the external filter plate 17, but flows via the external flow channel 14 to the liquid outlet 22, thus leaving the column 1 untreated. In an alternative embodiment, such liquid outlet 22 may be absent and the outer flow channel 14 may be sealed at its axial end remote from the space 19, for example by connecting the wall 18 to the axial housing end plate 3.

The bottom end of the filter bed 7 contains a centrally located closable packing port 9 to provide packing material for column packing purposes to the space between the filter plates 16, 17. For example, if the filter bed 7 is filled with material in a different manner, the filling port 9 and associated seals and components 10 and 11 may be absent.

O-ring seals are used to seal the core to the axial end plates and the filter bed and to seal the cover to the axially extending housing wall.

Fig. 7a shows a plain weave (PL), fig. 7b shows a twill weave (TL), fig. 7c shows a plain netherlands weave (PDW), and fig. 7d shows a twill netherlands weave (TDW). Fig. 8a shows a reverse plain netherlands weave (PZ), fig. 8b shows a reverse twill netherlands weave (KPZ), and fig. 8c shows a five-harness weave (FHD). Fig. 9 shows four layers (top to bottom: protective layer, filter layer, distribution layer, single reinforcement layer), fig. 10 shows five layers (double reinforcement layer), and fig. 11 shows a six layer (triple reinforcement layer) filter plate. In contrast to the filter plates of fig. 9-11, the filter plates of the present invention are obtained by: in the case of fig. 9, by eliminating one or both of the topmost and bottommost layers (as seen in the figure). In the case of fig. 10 or 11, by eliminating at least one of the top layer (protective layer) and one, two or all of the reinforcement layers. Figure 12 shows a bed section taken from a toroidal bed filter.

The column operates as follows: fluid is introduced into the distribution space through the supply channel and flows radially outwardly therefrom to the inlet channel. In the inlet channel, the fluid flows axially downwards to be evenly distributed over the entire surface of the outer filter plate. The fluid then flows radially inward through the packing to the inner filter plate through the outer filter plate. The fluid then flows through the inner filter plate uniformly distributed over the entire surface of the inner filter plate into the outlet channels. The fluid flows axially down the outer surface of the wick through the outlet channel and is collected in the collection space. From there, the fluid flows into the discharge channel. If the core contains a vent passage, for example as shown in FIG. 5, fluid flows axially upward through the core.

Further embodiments are also covered by the appended claims. For example, the flow direction of the incoming fluid may be reversed, for which purpose the supply, discharge, inlet and outlet elements are interchanged. Different embodiments also belong to the invention. The different features of the embodiments disclosed herein may be combined in different ways and different aspects of some features are considered interchangeable with each other. All the features described or disclosed in the figures provide the subject-matter of the invention equally or in any combination, also independently of their arrangement in the claims or their references. The figures, description and claims contain many combined features. The skilled person will consider these separately and combine them into further embodiments.

Conclusion: preferably, the liquid chromatography column, with a horizontal or radial flow of sample material passing therethrough, preferably in an inward direction, comprises: a housing defining a chamber therein; first and second axially or longitudinally extending porous filter plates located within the chamber of the housing; a bed or packing of chromatographic separation material, preferably in particulate form, located within said chamber of said housing and intermediate said porous filter plates, a first of said porous filter plates being adjacent said housing and external flow channels and a second of said porous filter plates being adjacent an optional core member and internal flow channels; the bed is in a circular ring shape; dispensing means operatively connected to said external flow passage; collecting means operatively connected to the inner flow channel, the distributing means and the outer flow channel being configured to guide the relevant material to be separated uniformly in the bed in a substantially horizontal direction across the longitudinal length of the bed, preferably the porous filter plates being coaxially positioned relative to each other, the first porous filter plate having a larger cross section than the second porous filter plate and the core member being located in the centre of the housing chamber.

Claims (9)

1. A liquid chromatography column designed to be radially flowed through by a process liquid and comprising beads between 120 and 1 millimeter diameter and which are hydrophilic packed beds, the beads being designed to capture biological products from mammalian CHO cells or cell cultures or cell fermentation harvests of 5-20 micrometer diameter from the process liquid, the packed beds of beads being held between an outer filter plate and an inner filter plate of the column and the packed beds being annular, wherein the outer filter plate has a first surface area and the inner filter plate has a second surface area smaller than the first surface area; the ratio of the first surface area to the second surface area is equal to an inlet filter plate surface area/outlet filter plate surface area (I/O) of the column and less than 5:1, such that the first surface area is no greater than 5 times the second surface area; the outer and inner filter plates have hydrophilic surfaces and each comprise at least two layers or sheets of stainless steel braided wire directly overlying each other on opposite surfaces, or the outer and inner filter plates are made of stainless steel and have a thickness of at least 0.3mm, the outer filter plate being adjacent to an axially extending housing wall of the column, the inner filter plate being disposed closer to a central longitudinal axis of the column;

A core member disposed in a space defined by the inner filter plate, the core member having an outer wall defining an inner flow channel with the inner filter plate, the inner flow channel having a width of at least 0.5mm, the inner flow channel tapering along the core member in an axial direction from one end to an opposite end of the post; and

the column is designed such that the liquid to be treated is first distributed over the surface of the outer filter plate, then passes through the outer filter plate, then flows radially inwards through the packed bed to the inner filter plate, then passes through the inner filter plate to enter the inner flow channel and then flows axially along the core member and finally is discharged from the column.

2. The column of claim 1, wherein the filtration layers of the outer and inner filter plates are directly exposed to the annular packed bed.

3. The column of claim 2, the filtration layers of the outer and/or inner filter plates being woven according to a plain netherlands weave.

4. The column of claim 1, inlet filter plate surface area/outlet filter plate surface area (I/O) being at least 1.5:1 and no greater than 4:1.

5. The column of claim 1, the packed bed height being at least 10 millimeters and/or no greater than 200 millimeters.

6. The column of claim 1, the packed bed volume being at least 10 milliliters and/or no greater than 20 liters.

7. The column of claim 1, the diameter of the inner filter plate being at least 10 mm and/or less than 150 mm.

8. The cartridge of claim 1, an outer flow channel defined by an outer filter plate and a housing wall of the cartridge having a width of at least 0.5 millimeters, and the inner flow channel having a width equal to the outer flow channel width times an inlet filter plate surface area/outlet filter plate surface area (I/O).

9. The column of any one of claims 1-8, the inner and outer filter plates being made of exactly three layers of braided wire of stainless steel directly superimposed on each other and providing a pore size of at least 100 microns, a packed bed height of at least 20 mm and below 150 mm, hydrophilic beads having a size between 200 microns and 1 mm.